Human urokinase is an enzyme found as a trace in human urine capable of activating inactive plasminogen into plasmin, and the plasmin thus formed is capable of dissolving fibrin. This urokinase consists of two polypeptide chains linked together by a disulfide bond. On the contrary, human prourokinase is a single chain polypeptide in which the aforesaid two polypeptide chains are joined together through an amide bond. Although this prourokinase itself does not possess the aforementioned activity, it can be converted to the preceding active urokinase by cutting one amide bond.
Japanese Patent Application No. 61-12984 (Japanese Unexamined Patent Publication No. 62-143686) (EP 0210279) discloses stabilized human prourokinase-like polypeptides wherein the 135th amino acid and the 157th amino acid are varied, but these are intended to inhibit a cleavage between the 135th amino acid and the 136th amino acid, and/or a cleavage between the 158th amino acid and the 159th amino acid, and therefore, are completely different from the polypeptides of the present invention.
Both the prourokinase described in EP 0200451 and the prourokinase-like polypeptides described in Japanese Patent Application No. 61-12984 (Japanese Unexamined Patent Publication No. 62-143686) (EP 0210279) do not practically exhibit the activities thereof at a site at which thrombus is present, and therefore it is assumed that, even though applied in a large amount, side effects such as systemic hemorrhage are prevented. Nevertheless, it is considered that the conversion rate thereof to a two chain type is low.
As described above, the human urokinase is an activated enzyme, and the activity thereof is rapidly lost due to the presence of a large amount of various inhibitors in blood, and therefore, where used as a therapeutic agent, a large amount thereof must be administered. As a result, a side effect of a systemic formation of plasmin occurs, which tends to cause systemic hemorrhage.
Although prourokinase, also called single-chain urokinase, is usually inactive in plasma, it exhibits a weak activity at a site at which thrombus is present, to convert plasminogen to plasmin. It is considered that, since a small amount of plasmin produced by the action of a tissue plasminogen activator or prourokinase converts prourokinase to a two-chain type high molecular weight urokinase having a high activity, the conversion of plasminogen to plasmin rapidly proceeds, and the thrombus is lyzed (literature 1).
Where prourokinase is used as thrombolytic agent, a small application amount does not produce a sufficient amount of plasmin, and therefore, the thrombus cannot be efficiently lyzed. Conversely, in the case of an excess application amount, even though a large amount of plasmin is temporarily produced, an increased amount of the plasmin does not increase the thrombolysis, due to the distance thereof from a thrombus site, resulting in a decreased thrombolysis efficiency. Moreover, a portion of plasmin thus produced is inactivated by inhibitors such as .alpha..sub.2 -antiplasmin, and another portion activates prourokinase at a site at which thrombus is not present, resulting in side effects such as systemic hemorrhage. Therefore, when administering prourokinase, a dose and manner of administration which will not produce a temporal excess amount of plasmin is described. Moreover, it is reported that prourokinase, when cleaved at a peptide bond between Arg 156 and Phe 157, is no longer activated by plasmin (literature 2), and accordingly, the prourokinase cannot properly exhibit its function in the presence of thrombin.
EP 0200451 describes protease-resistant urokinases wherein the 156th to 158th amino acids are changed, but all of these urokinases are intended only to prevent protease cleavage at a site between the 158th amino acid and the 159th amino acid, on the assumption that the cleavage of this site is not necessary when activating prourokinase, and accordingly, are completely different from the present polypeptides.
Accordingly, a new type of human prourokinase is sought which, while maintaining a high thrombus specificity of human prourokinase, does not exhibit side effects due to a large application amount, is rapidly activated during and after the formation of thrombus, and therefore, does not have the above-mentioned disadvantages of human urokinase, human prourokinase, and aforementioned derivatives thereof. Such a human prourokinase is not known.